Tyrosine phosphorylation of CARM1 promotes its enzymatic activity and alters its target specificity

An important epigenetic component of tyrosine kinase signaling is the phosphorylation of histones, and epigenetic readers, writers, and erasers. Phosphorylation of protein arginine methyltransferases (PRMTs), have been shown to enhance and impair their enzymatic activity. In this study, we show that the hyperactivation of Janus kinase 2 (JAK2) by the V617F mutation phosphorylates tyrosine residues (Y149 and Y334) in coactivator-associated arginine methyltransferase 1 (CARM1), an important target in hematologic malignancies, increasing its methyltransferase activity and altering its target specificity. While non-phosphorylatable CARM1 methylates some established substrates (e.g. BAF155 and PABP1), only phospho-CARM1 methylates the RUNX1 transcription factor, on R223 and R319. Furthermore, cells expressing non-phosphorylatable CARM1 have impaired cell-cycle progression and increased apoptosis, compared to cells expressing phosphorylatable, wild-type CARM1, with reduced expression of genes associated with G2/M cell cycle progression and anti-apoptosis. The presence of the JAK2-V617F mutant kinase renders acute myeloid leukemia (AML) cells less sensitive to CARM1 inhibition, and we show that the dual targeting of JAK2 and CARM1 is more effective than monotherapy in AML cells expressing phospho-CARM1. Thus, the phosphorylation of CARM1 by hyperactivated JAK2 regulates its methyltransferase activity, helps select its substrates, and is required for the maximal proliferation of malignant myeloid cells.

(B) Anti-CARM1-Y334 phosphospecific antibody recognized CARM1 in the presence of active JAK2 kinase in a dose dependent manner (left).Anti-CARM1-Y149 phosphospecific antibody recognized CARM1 in the presence of active JAK2 kinase in a dose dependent manner (right).In both assays, the complete abolishment of CARM1 phosphorylation by JAK1/JAK2 inhibitor (ruxolitinib, RUX; 500 nM) was observed.
(E) Endogenous phosphorylated CARM1 was immunoblotted from total cell lysates of HEL cells treated with DMSO or RUX, using anti-CARM1-Y149 (left) and -Y334 (right) phosphospecific antibodies.The reduction of CARM1 phosphorylation was observed at the higher RUX concentration (500 nM).
(F) Phosphorylation of Y334 of CARM1 in HEL cells is reduced following treatment with the highly selective JAK2 inhibitor (NVP-BSK805, 6 μM).

Supplementary Figure 6. Dimerization and subcellular localization of CARM1
(A) HA-tagged WT-CARM1 and MYC-tagged WT-CARM1 constructs were co-transfected into 293T cells.Following immunoprecipitation with anti-MYC antibody, the detectable HA-tagged WT-CARM1 was decreased in the 293T cells treated with RUX 10 μM vs. those treated with DMSO (RUX 0 μM). the darker color of the node indicates the more statistically significant P-value.CARM1-associated proteins clustered into groups of proteins identified as being involved in purine nucleotide metabolism, E2F targets, MYC targets, histone and chromatin binding proteins, and ribonucleoprotein complex biogenesis, based on enriched pathway analysis of Metascape (metascape.org) for both cell lines.(D) Proteins binding to CARM1 and enrichment network visualization for the results in HEL and K562 cells.See also supplementary data.As JAK2 are weakly and transiently associated with its substrates, we did not observe the interaction of CARM1 with JAK2 in HEL cells.
(E) Targeted mass spectrometry analysis using parallel reaction monitoring revealed the interaction of CARM1 with RUNX1 in HEL cells.Skyline chromatogram visualizations showed the intensity at each resampled retention time point for all fragment ions, which were presented as different colored lines identified in the legends.Peak areas by each fragment were displayed as the bar graphs (left) to compare the intensity of samples from HEL cells with CARM1-BirA* fusion (total AUC value, 395,628) to those with BirA* alone (total AUC value, 215,928).
(C)(D)(E) All error bars represent the mean ± SD.P values were determined by t one-way ANOVA followed by Dunnett's post hoc test.

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B) HA-tagged WT-CARM1 and MYC-tagged WT-CARM1 constructs were co-transfected into 293T cells overexpressing HA-tagged WT-JAK2 or JAK2-V617F mutants.Following immunoprecipitation with anti-HA antibody, the detectable MYC-tagged WT-CARM1 was increased in the 293T cells expressing JAK2-V617F (lane 16) vs. those expressing WT-JAK2 (lane 15).(C) The immunoblotting assay of subcellular localizations in HEL, SET2, and K562 cells revealed that total CARM1 is localized mainly in the cytoplasm.Purity of the subcellular fractions was assessed by detecting HSP90 (cytoplasmic extraction, CYE), SP1 (nuclear soluble extraction, NSE), and histone H3 (chromatin-bound extract, CBE).WCE indicates proteins within the whole-cell lysates.(D) Immunoblotting to assess CARM1 subcellular localization in HEL cells treated with DMSO or ruxolitinib (RUX) (500 nM).Supplementary Figure 7.The interactome of CARM1 by BioID system (A) Schematic diagram demonstrating BioID (proximity-dependent biotin identification) approach for the identification of CARM1-interacting proteins.(B) Western blots confirming biotinylated proteins of lysate from HEL cells with CARM1-BirA* fusion proteins overexpression in culture medium with or without biotin.(C) Metascape enrichment network visualization showing the intra-cluster and inter-cluster similarities of enrichment terms, up to nine terms per clusters, in HEL cells.Terms are defined according to GO/KEGG terms, canonical pathways, and hallmark gene sets.The connecting pairs of nodes are created with Kappa score >0.3.Terms containing more genes tend to have a more significant P-value;